ABSTRACT A paradigm-shift in immunogerontology has been occurring by finding that TLR signal is attenuated in elders, suggesting that an alternative pro-inflammatory mechanism may be engaged in the age-associated periodontitis. Ceramides that compose the cell membrane of vertebrates are attracting interests as signaling mediators in a variety of cell events, including, apoptosis and cell senescence. We recently reported that intracellular ceramides promote osteoclastogenesis by acting on a non-muscle myosin IIA (Myh9), an intracellular down-regulatory factor for cell fusion event in osteoclastogenesis. On the othernhad, it was recently demonstrated that intracellular ceramides directly act on cathepsin B to activate its catalytic activity. It is known that cathepsin B promotes RANKL-mediated osteoclastogenesis via temporally controlled proteolysis of Myh9 for initiation of cell fusion. In response to aging, the amount of ceramides increases in the cytoplasm of cells in conjunction with the diminished level of acid ceramidases (aCDase), a lipid hydrolase that degrades ceramides. Our preliminary results indicate that high mobility group protein B1 (HMGB1), a key senescence-associated proinflammatory mediator, downregulated aCDase expression in Raw264.7 cells. Based on these lines of evidence, we hypothesize that in the context of age associated periodontitis, where the LPS-induced signaling appears to be diminished, HMGB1 increases intracellular ceramides to advance OC-mediated bone loss by acting on cathepsin B, a promoter of osteoclastogenesis. In this R15 project, the Specific Aim 1 will establish the role of HMGB1 in accumulation of intracellular ceramide of senescence OCPs via downregulation of aCDase activity. We will compare the role of HMGB1 and LPS isolated from either Porphyromonas gingivalis or Escherichia coli relative to the downregulation of aCDase activity and resulting accumulation of ceramides in cytoplasm of OCPs of young (two month-old) and aged (twenty four month-old) wild type mice, both in vitro osteoclastogenesis assays and in in vivo mouse model of periodontitis. In Specific Aim 2, we will investigate the impact of intracellular ceramides on cathepsin B-dependent upregulation of osteoclastogenesis. We will evaluate the effects of intracellular ceramides on upregulation of cathepsin B's catalytic activity that induce cells fusion event in osteoclastogenesis via Myh9 proteolysis in vitro. The proposed research project will, for the first time, investigate the possible pathogenic role of intracellular ceramide in age-dependently elevated osteoclastogenesis and will provide a foundation for the development of novel therapeutic approach for periodontitis in aging population. The most importantly, the proposed R15-AREA project will provide a unique opportunity for undergraduate students to participate in the biomedical researches at one of the largest academic Hispanic-serving institutions of Nova Southeastern University.